The invention relates to a process for preparing 3-cephalosporin C derivatives which are used in the preparation of xcex2-lactam antibiotics. In particular the invention relates to an enzymatic process for the preparation acid (3-thiolated-7-ACA) using xcex1-ketoacid intermediates. The xcex1-ketoacids or xcex1-oxoacids are important biopharmaceutical compounds.
Oxoacids of essential amino acids are gaining importance as nutraceuticals (Pszcola, D E, Food Technol. 52, 30, 1998) as well as therapeutic agents for treating nitrogen accumulation disorders (Schaefer et al., Kidney Int. Suppl. 27, S136, 1989; Buto et al, Biotechnol. Bioeng. 44, 1288, 1994). Another important application is the production of 7-amino cephalosporanic acid (Savidge, T A; In Biotechnology of Industrial Antibiotics, p 171, Marcel Dekker, New York, 1984) from cephalosporin C (3-acetoxymethyl-7xcex2-(D-5-amino-5-carboxypentanamido) ceph-3-em-4-carboxylic acid). The transformation can be carried out by a D-amino acid transaminase from Bacillus licheniformis ATCC 9945, which converts cephalosporin C with xcex1-ketoacids into xcex1-ketoadipyl-7-ACA and the corresponding D-xcex1-amino acid, as described in DE 3447023 (Hoechst). This conversion is a transamination, the amino group of cephalosporin C being converted non-oxidatively into the keto group, without the release of hydrogen peroxide. However there is a low level of activity of this enzyme, as described in EP 0315786.
Chemical methods for the preparation of 3-thiolated-7-ACA cephalosporanic acid derivatives are known (U.S. Pat. No. 3,367,933; BE 718,824), however they have disadvantages such as low temperature reaction conditions, the use of costly and toxic solvents or reagents and chemical instability of intermediates which makes the processes difficult on an industrial scale.
To overcome the drawbacks of the chemical route to 7-ACA, alternative enzymatic cleavage of cephalosporin C has been described. Direct one-step removal of the lateral 7xe2x80x2-aminoadipic side-chain of cephalosporin C is possible by using specific cephalosporin acylases (FR 2,241,557; U.S. Pat. No. 4,774,179; EP 283,248; WO 9512680; WO 9616174). These processes, however, are often not reproducible and are characterised by low yields and lengthy reaction times as described in U.S. Pat. No. 5,296,358. No industrial application of this technology (single-step conversion of cephalosporin C to 7-ACA) has been reported at this time (Parmar et al, Crit. Rev. Biotechnol. 18, 1, 1998).
On the other hand, processes that transform the cephalosporin C into 7-ACA by means of two enzymatic steps are important from an industrial point of view. The first stage consists of using a D-amino acid oxidase (E.C. 1.4.3.3, hereinbelow indicated as DAAO) from different sources (Trigonopsis variabilis, GB 1,272,769; Rhodotorula gracilis, EP 0,517,200; or Fusarium solari M-0718, EP 0,364,275). DAAO oxidises the lateral D-5-amido-carboxypentanoyl chain of cephalosporin C in the presence of molecular oxygen, to produce 7xcex2-(5-carboxy-5-oxopent-amido)-ceph-3-em-carboxylic acid (or xcex1-ketoadipyl-7-aminocephalosporanic acid, hereinbelow indicated as xcex1-ketoadipyl-7-ACA) and hydrogen peroxide, which chemically decarboxylate the xcex1-ketoadipyl-7-ACA to 7xcex2-(4-carboxy butanamido)-ceph-3-em-4-carboxylic acid (or glutaryl-7-aminocephalo-sporanic acid, hereinbelow indicated as GL-7-ACA).
In a second stage, a specific acylase for GL-7-ACA, glutaryl-7-ACA acylase (E.C. 3.5.1.3), is used, for example that of a Pseudomonas type microorganism (U.S. Pat. No. 3,960,662, EP 0496993) over expressed in E. coli, which deacylates the GL-7-ACA into 7-amino-ceph-3-em4-carboxylic acid (or 7-amino cephalosporanic acid, hereinbelow indicated as 7-ACA).
This two-step enzymatic process for obtaining 7-ACA has been used on an industrial scale (Conlon et al. Biotechnol. Bioeng. 46, 510,1995).
Yet another advance in enzymatic processes, is disclosed in EP 0846695, in which solid glutaryl-7-ACA is reacted with a heterocyclic group that contains at least a nitrogen with or without a sulphur or oxygen atom to produce a 3-modified glutaryl-7-ACA. These 3-derivatives are enzymatically transformed to their corresponding 3-heterocyclic thiomethyl-7-ACA derivatives.
This procedure can be defined as an enzymatic-chemical-enzymatic (ECE) process, since the isolated GL-7-ACA comes from a bioconversion of solubilised cephalosporin C, then GL-7-ACA is reacted with the heterocyclic thiols and finally the 3-heterocyclic thio-derivative is enzymated with GL-7-ACA acylase. The problem with this method is the need to isolate GL-7-ACA, which given its high water solubility, is technically difficult and expensive, as described in WO 9535020.
An additional problem is that the enzyme can only be reused a few times due to the xe2x80x9cpoisoningxe2x80x9d of the biocatalyst by the residual heterocyclic thiols. This poisoning effect is well documented with one of the thiols used, 5-methyl-1,3,4-thiadiazole-2-thiol (MMTD) (Won et al, App. Biochem. Biotech. 69, 1, 1998).
The oxidative deamination of the D-adipamido side chain of cephalosporin C under aerobic conditions into xcex1-ketoadipyl-7-ACA has been described using D-amino acid oxidase (D-AAO) from cell-free extracts (GB 1,2 72,769, Glaxo) or in toluene-activated (permeabilised) cells (GB 1,385,685) of the yeast Trigonopsis variabilis or Rhodotorula glutinis (EP 0517200). In this reaction, molecular oxygen acts as the electron acceptor and is converted to hydrogen peroxide, which chemically reacts with the xcex1-ketoadipyl-7-ACA producing its decarboxylation into glutaryl-7-ACA. In the presence of large quantities of the catalase produced for the above yeasts, the hydrogen peroxide is cleaved to water and molecular oxygen, rendering a mixture of xcex1-ketoadipyl-7-ACA and glutaryl-7-ACA. The xcex1-ketoadipyl-7-ACA is quite unstable (GB 1,385,685) and rapidly decomposes to unknown products and hence reduces the yield of glutaryl-7-ACA from 90 to 95% to 60 to 70%, depending on the yeast and strain (Parmar et al, Crit. Rev. Biotechnol. 18, 1, 1998; Rietharst, W. and Riechert, A, Chimia 53, 600, 1999). As a result no industrial application has been described.
There is therefore a need for an efficient and improved process for the preparation of 3-thiolated-7-ACA cephalosporanic acid derivatives on an industrial scale. In addition the isolation of stable xcex1-ketoacid derivatives which are important biopharmaceutical compounds would be beneficial.
According to the invention there is provided a process for preparing cephalosporanic acid derivatives comprising the steps of:
enzymatically converting a 3-thiolated cephalosporin C compound of formula III: 
into a 3-thiolated-xcex1-ketoadipyl-7-aminocephalosporanic acid derivative of formula IV: 
wherein R is a heterocyclic group comprising at least a nitrogen atom.
Preferably the compound of formula III is enzymatically converted into a compound of formula IV by an immobilised enzyme system. Most preferably the enzyme system comprises co-immobilised D-Amino acid oxidase and catalase.
Preferably the enzymatic conversion is carried out in the presence of molecular oxygen, at a pressure of 1 to 5 bar absolute, a pH of from 6.5 to 8.0 and at a temperature of from 15 to 30xc2x0 C. for a period of from 30 mins to 180 mins.
Preferably the process comprises the step of separating the enzyme system from the reaction mixture, preferably by filtration.
In one embodiment of the invention the process includes the step of purifying the compound of formula IV.
Most preferably the compound is purified using an adsorption column. Preferably the enzymes are co-immobilised using a suitable cross-linker agent in a suitable solid support. The enzymes may be in the form of crystals of a size suitable for use as a biocatalyst.
Preferably the enzymatic processes are carried out while maintaining the enzyme in dispersion in an aqueous substrate solution. Preferably the or each enzymatic process is carried out in a column. Most preferably the process includes the step of recovering the enzyme for reuse.
In one embodiment of the invention the compound of formula IV is used without purification in a continuous process for obtaining any useful derivative.
Preferably the R group in compounds of formula III and IV is a heterocyclic group comprising at least one nitrogen atom and optionally a sulphur or oxygen atom.
Most preferably R is a heterocyclic group selected from any one or more of the group comprising thienyl, diazolyl, tetrazolyl, thiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzo thiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably 5-methyl-1,3,4-thiadiazol-2-yl, 1-methyl-1H-tetrazol-5-yl or 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
The invention provides a 3-thiolated-xcex1-ketoadipyl-7-aminocephalosporanic acid derivative of formula IV whenever prepared by a process of the invention.
The invention provides a compound of the Formula: 
wherein in formula IV, R is 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
The invention provides a compound of the Formula: 
wherein in formula IV, R is 1-methyl-1H-tetrazol-5-yl.
The invention also provides use of a compound of formula IV as an intermediate in a process for preparing cephalosporin C antibiotics.
The invention also provides use of an intermediate compound of the formula: 
in a process for preparing cephalosporin C antibiotics wherein in formula IV R is 5-methyl-1,3,4-thiadiazol-2-yl.
The invention further provides a process for preparing cephalosporanic acid derivatives of the invention comprising the step of:
enzymatically converting a compound of formula IV to form a compound of formula I 
wherein R is a heterocyclic group comprising at least one nitrogen atom and R1 and R2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
Preferably the compound of formula IV is enzymatically converted to form a compound of formula I using Glutaryl-7-ACA acylase, most preferably the enzymation takes place at a temperature of approximately 20xc2x0 C. and at a pH of between 6.5 and 8.0. Preferably the enzyme is immobilised using a suitable cross-linker agent in a suitable solid support.
Preferably the enzyme is in the form of crystals of a size suitable for use as a biocatalyst.
In one embodiment of the invention the enzymation is carried out while maintaining the enzyme in dispersion in an aqueous substrate solution. Preferably the enzymatic process is carried out in a column. Most preferably the process includes the step of recovering the enzyme for reuse.
The invention also provides use of a compound of formula I as an intermediate in a process for preparing cephalosporin C derivatives.
The invention further provides a process for preparing 3-thiolated cephalosporanic acid derivatives comprising the steps of;
enzymatically converting a compound of formula III 
into a 3-thiolated-xcex1-ketoadipyl-7-aminocephalosporanic acid derivative of formula IV: 
and enzymatically converting a compound of formula IV to form a 3-thiolated 7-ACA compound of formula I 
wherein R is a heterocyclic group comprising at least one nitrogen atom and R1 and R2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
In one embodiment of the invention the compound of formula III is enzymatically converted into a compound of formula I in one step by an immobilised enzyme system. Most preferably the enzyme system comprises a combination of co-immobilised D-amino acid oxidase/catalase in the presence of immobilised Glutaryl-7-ACA acylase. Preferably the enzymation takes place at a temperature of approximately 20xc2x0 C. and at a pH of between 6.5 and 8.0. Most preferably the enzymes are co-immobilised using a suitable cross-linker agent in a suitable solid support.
Preferably the enzymes are in the form of crystals of a size suitable for use as a biocatalyst.
Most preferably the enzymatic processes are carried out while maintaining the enzyme in dispersion in an aqueous substrate solution.
Preferably the or each enzymatic process is carried out in a column. Most preferably the process includes the step of recovering the enzyme for reuse.
In one embodiment of the invention the compound of formula III is used without purification in a continuous process for obtaining any useful derivative.
The invention also provides a process for preparing cephalosporanic acid derivatives comprising the steps of:
reacting cephalosporin C with a thiol compound of the general formula II
Rxe2x80x94SHxe2x80x83xe2x80x83II 
wherein R is a heterocyclic group comprising at least one nitrogen atom,
to form a 3-thiolated cephalosporin compound of formula III 
wherein R is as defined above,
and, after formation of the compound of formula III removing excess thiol of formula II.
In one embodiment of the invention the excess thiol is removed by adsorption on an anion exchange resin. Preferably the anion exchange resin is a microporous resin having a cross-linked acrylic copolymer structure. Most preferably the anion exchange resin comprises an 8% cross-linking containing functional thialkyl benzyl ammonium group. The resin may be in the chloride, hydroxy, phosphate or acetate cycle.
In another embodiment of the invention the excess thiol is removed by crystallisation. Preferably crystallisation is carried out at an acidic pH.
In a further embodiment of the invention the excess thiol is removed by crystallisation followed by adsorption on an anion exchange resin.
Preferably the cephalosporin C is in an aqueous medium, Most preferably the cephalosporin C is in the form of a concentrated cephalosporin C solution.
Preferably the reaction is carried out at a pH of between 5.5 and 8.0, at a temperature of from 60xc2x0 C. to 80xc2x0 C., for a period of from 1 to 8 hours. Most preferably the reaction is carried out at a pH of approximately 6.0 and at a temperature of approximately 65xc2x0 C.
In one embodiment of the invention the thiol compound is present in an amount of between 1 and 5 mol/mol of cephalosporin C.
Preferably R is a heterocyclic group comprising at least one nitrogen atom and optionally a sulphur or oxygen atom. Most preferably R is a heterocyclic group selected from any one or more of thienyl, diazolyl, thiazolyl, tetrazolyl, thiadiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzothiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably 5-methyl-1,3,4-thiadiazol-2-yl, 1-methyl-tetrazol-5-yl or 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
The invention provides a compound of formula III 
wherein R is a heterocyclic group comprising at least one nitrogen atom,
The invention provides a compound of the formula: 
wherein in formula III R is 5-methyl-1,3,4-thiadiazol-2-yl.
The invention further provides a compound of the formula: 
wherein the formula III R is 1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl.
The invention also provides use of a compound of formula III as an intermediate in a process for preparing cephalosporin C derivatives.
One embodiment of the invention provides a process for preparing cephalosporanic acid derivatives comprising the steps of:
enzymatically converting a 3-thiolated cephalosporin C compound of formula III obtained by a process as described above: 
into a 3-thiolated-xcex1-ketoadipyl-7-aminocephalosporanic acid derivative of formula IV: 
wherein R is a heterocyclic group comprising at least a nitrogen atom.
In one embodiment of the invention the process additionally comprises the step of:
enzymatically converting a 3-thiolated xcex1-ketoadipyl 7-ACA compound of formula IV 
to form a 3-thiolated 7-ACA compound of formula I 
wherein R is a heterocyclic group comprising at least one nitrogen atom and R1 and R2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
Another embodiment of the invention provides a process for preparing cephalosporanic acid derivatives comprising the step of:
enzymatically converting a compound of formula IV 
to form a compound of formula I 
wherein R is a heterocyclic group comprising at least one nitrogen atom and R1 and R2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
Preferably a compound of formula IV is enzymatically converted to form a compound of formula I with Glutaryl-7-ACA acylase.
Most preferably compounds of formula I, III and IV are in a solid form or in the form of a non-toxic salt thereof.
The invention provides a process for the preparation of cephalosporin C antibiotics and derivatives thereof comprising forming a compound of formula III, IV and I as hereinbefore defined and subsequent enzymation of the compound.
The antibiotic may be any one or more of cefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiam and ceftriaxone.
We have found that 3-thiolated cephalosporanic C derivatives may be enzymated into xcex1-ketoacid derivatives in the presence of a co-immobilised D-amino acid oxidase/catalase system. These xcex1-ketoacid derivatives have been shown to be stable when isolated. A new improved process to obtain 3-thiolated-7-ACA derivatives, both in one step or in two consecutive enzymatic steps is thereby provided.
The present invention relates to an improved and more efficient process for preparing compounds of formula I from cephalosporin C. 
wherein R is a heterocyclic group comprising at least one nitrogen atom and R1 and R2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
The process involves the formation of new stable xcex1-ketoadipyl-7-ACA derivative intermediates. Alternatively the process may be carried out in a single one pot reaction without the formation of intermediates.
It was surprisingly found that the 3-thiolated derivative of cephalosporin C were very good substrates for the enzymatic reaction by D-amino acid oxidase in the presence of catalase.
3-thiolated-cephalosporin C derivatives of formula III 
are prepared from cephalosporin C. The cephalosporin C solution may be in a purified or crude form. The cephalosporin C is in the form of any non-toxic salt of cephalosporin C.
The reaction of nucleophilic substitution in the 3xe2x80x2 position is carried out in an aqueous medium, dissolving the heterocyclic thiol and any non-toxic cephalosporin C salt in water by addition of a basic compound which form a water soluble salt, such as alkali metal hydroxide, ammonium hydroxide or preferably alkali metal carbonate or bicarbonate. In general, in addition to salts produced as described above, any commercially available salt of cephalosporin C and of the heterocyclic thiols can be used in the process of this invention without changing the fundamentals of the process.
After dissolving the heterocyclic thiol and the cephalosporin C, in separate reaction vessels or jointly, both reactants are mixed together in the same reactor, before or after heating the solution to a temperature from about 65xc2x0 C. to 80xc2x0 C. at a pH value of between 5.5 and 7.0.
Once the reaction starts, the temperature and pH are maintained preferably at approximately 65xc2x0 C. and 6.0 respectively, for a period of time of approximately 1 hour to 4 hours.
The heterocyclic thiol/cephalosporin C molar ratio is an important variable in the yield of the reaction and has to be optimised for each heterocyclic thiol used. Molar ratios are between 1.0 and 4.0, preferably at a molar ratio of approximately 4.
It was found that at these molar ratios the cephalosporin C remains quite stable with low xcex2-lactam ring degradation, compared to a cephalosporin C solution without the thiol, which is completely degraded within 40 min at 80xc2x0 C.
Once the cephalosporin C level is below 2% of the initial amount, the reaction mixture is cooled to a temperature from about 2xc2x0 C. to about 10xc2x0 C., with or without acidification at a pH of from pH 3.0 to 5.5, preferably approximately 5.2, with strong mineral acids, such as hydrogen halides or oxy acids.
This acidification step gives in some cases, crystallisation of the heterocyclic thiol, with the concomitant possibility of reuse for a new reaction.
After formation of the compound of formula III excess thiol groups are selectively removed which allows cephalosporin C derivatives of formula III to be prepared at very high purity levels and with very low levels ( less than 0.2 mg/ml) of heterocyclic thiols present. A highly selective removal procedure with the strong anion exchanger Amberlite IRA-400 (manufactured by Rohm and Haas) is utilised. This process has several advantages. It allows compounds of Formula III to be used as a substrate without the poisoning of the enzymes in the next process step. As a result the enzymes may be used repeatedly. In addition the process does not require the use of toxic reagents or the need to isolate intermediates thereby providing a continuous process.
Different resins and types of chromatography may be used on an industrial scale.
Several resins were tested grouped in four classes of resins based on adsorption, hydrophilic-hydrophobic interaction, cation exchange, and anion exchange. All resins tested based on adsorption (Amberlite XAD-761, Amberlite 7HP, Amberlite 16 HP and Amberlite XAD-4) gave similar results, the eluate containing from 22% to 38% of the heterocyclic thiol. The hydrophobic-hydrophilic interaction resin Sephadex LH-20 did not retain any thiol ( less than 5%). A similar situation was found with the cation exchangers Amberlite(copyright) IRC-50, IR-120 and IR-200. However, anion exchangers were found to have the best binding capacity for heterocyclic thiols ranging from 57-60% in the case of a weak anion exchanger (Amberlite IRA-93).
It was found that a strong microporous (gel-type I) anion (base) exchange resin Amberlite IRA-400 having an 8% cross linking containing function trialkyl benzyl ammonium groups gave the highest binding of heterocyclic thiols (from 92-98%) and low binding of the 3xe2x80x2-position heterocyclic thiomethyl cephalosporin C derivative (from 2-15%, less than 15% for the first cycle and less than 5% for the following cycles).
Such a microporous resin offers certain advantages. They are less fragile, require less care in handling and possess higher loading capacities. As they have no discrete pores solute ions diffuse through the particle to interact with exchange sites. The total exchange capacity of the mentioned resin is in the order of 1,4 meq/mL.
It was surprisingly found that Amberlite IRA-400 has less binding capacity for 3xe2x80x2-heterocyclic thiomethyl derivatives of cephalosporin C than for the same derivatives of glutaryl-7-ACA and 7-ACA. In fact the 3xe2x80x2-heterocyclic thiomethyl derivative of glutaryl-7-ACA produced with MND binds at a level of 76.3% to the column. The same result is found with 3xe2x80x2-heterocyclic thiomethyl derivative of 7-ACA, with MMTD, which binds at a level of 92.7% to the column. This unexpected behaviour of Amberlite IRA400 with these three related xcex2-lactam compounds appears to result from the presence of an ionisable amino group in the 5 position of the side chain of cephalosporin C compared with glutaryl-7-ACA and 7-ACA.
The removal of heterocyclic thiols by the process of the invention is particularly advantageous on an industrial scale as the eluate of the column can be used for enzymation without isolation of the modified cephalosporin C and represents a new concept in the field of cephalosporin intermediates wherein the impurities are bound to the column and the xcex2-lactam derivative is simply eluted by water.
Once the xcex2-lactam derivative is eluted (less than 5% remains bound), the column is typically regenerated with a 1.5 N solution of a strong mineral acid, such as hydrogen halide containing variable amounts of an organic solvent, preferably 10-20% acetonitrile. When the concentration of the thiol in the eluate is higher than 0.2 mg/ml, a strong regeneration using 3 N HCl and 40% acetonitrile may be carried out. Alternatively regeneration with 1.5M HCl and 1.0 N NaOH is also possible.
After elution of the heterocyclic thiol, the thiol is concentrated and reused. The column is rinsed with deionised water to remove excess regenerant before the next cycle. The first bed volume of the rinse should be performed at the flow rate used for regeneration. The remainder is run at the adsorption flow rate.
Compounds of formula III are enzymatically converted into new stable xcex1-ketoadipyl-7-ACA derivatives of formula IV by an immobilised enzyme system. 
wherein R is a heterocyclic group comprising at least a nitrogen atom with or without a sulphur or oxygen.
The use of co-immobilised enzymes (D-AAO and catalase) on the same solid support allows a better hydrogen peroxide removal than with separate supports. The biocatalyst with both enzymes is easily recoverable from the reaction medium and reusable a large number of times. This is a necessary and indispensable factor for an industrial process.
A further industrial advantage of the invention is the easy transfer of the chemical solution comprising a compound of formula III, after chromatography in a strong anionic exchange resin (Amberliteo(copyright) IRA-40, manufactured by Rohm and Haas) to an enzymatic reactor containing the co-immobilised enzymes. This enables the process to be conducted continuously with a single liquid stream from cephalosporin C to compound IV.
The enzymatic stage may be carried out in different ways:
1) Chemical reaction without removal of the excess of the heterocyclic thiol and oxidative deamination with immobilised D-AAO. Under these conditions, compound IV is accumulated to about 35 to 40% of total xcex2-lactams, which is higher than the 5 to 10% accumulation of xcex1-ketoadipyl-7-ACA produced when unmodified cephalosporin C is used. Thus indicating the stability of the compound IV.
2) The same as 1) including soluble catalase. Under these conditions, the level of compound IV reaches 70 to 75% of total xcex2-lactams in solution with less than 10% of 3-thiolated glutaryl-7-ACA (Txe2x80x2Xxe2x80x2G). However the immobilised enzyme and catalase are poisoned within a few cycles by the presence of high levels of heterocyclic thiol (xe2x96xa11 mg/mL).
3) Chemical reaction with removal of the excess of the heterocyclic thiol by ion exchange chromatography and co-immobilisation of D-AAO and catalase on the same solid support. Under these conditions, compound IV is accumulated from about 80% to 90% of total xcex2-lactams, depending on the pH used. At pH values of approximately 6.5, compound IV is more stable reaching 90% accumulation but the D-AAO is less active (more biocatalyst is needed). At pH values near 7.25, the enzyme is more active but compound IV is less stable, obtaining a 80% accumulation. The preferred pH is pH 6.75, which provides the lowest loss in D-AAO activity with good stability of compound IV.
From the above it is clear that approach 3) is advantageous versus the others but several parameters have to be taken into account to produce a good biocatalyst with the two enzymes (D-AAO and catalase) co-immobilised:
a) The source of both enzymes. D-AAO in this invention is obtained from Trigonopsis variabilis CBS 4091 obtained from the Spanish collection of microorganisms (CECT, Valencia, Spain). This yeast is grown under the conditions to induce D-AAO (Kubicek et al, J. Appl. Biochem. 7; 104, 1985) and the enzyme was purified by ammonium sulfate fractionation between 30%-55% as described by Szwjcer et al (Biotechnol. Lett. 7, 1, 1985). Amino acid oxidases may also be sourced from Rodotorula gracilis. Catalase from Micrococcus lisodeikticus is obtained from a commercial source (Fluka, Madrid, Spain), but it may also be sourced from Aspergillas niger. 
b) The solid support used. Several carriers are available to immobilise enzymes. The most popular are: Amberlite(copyright) IRA 900 (strongly basic polystyrene resin with a quaternary amine function), Duoliteo(copyright) A365 (weakly basic polystyrene resin with primary functional groups), Duoliteo(copyright) A568 (moderately basic poly-condensed phenolformaldehyde resin), BrCN-activated Sepharose(copyright), vinyl Sepharose(copyright) and Eupergit C(copyright) (based on a polyacrylic structure and in particular with oxirane terminal groups). Among Eupergit, two classes are commercialised (Rxc3x6hm Pharma) C and C250L, the latter type is particularly suitable for binding high molecular weight enzymes, since its contents in oxirane groups are at least 0.36% compared with the 0.93% in Eupergit C. This C250L type show outstanding properties when employed in industrial biocatalytic processes. The morphology of the carrier, i.e., its narrow particle size distribution (200 xcexcm) and high mechanical stability accounts for their good properties in stirred-tank reactors. It is not mechanically destroyed in stirred systems and filtration at the end of the reaction cycle is quick and very easy to perform. Changes in the pH and ionic strength have no effect on swelling of the matrix. In addition, this Eupergit C250L has never been used to immobilise D-amino acid oxidase and catalase.
c) The ratio catalase units/D-AAO units. This ratio is normally bigger than 100, but for efficient hydrogen peroxide removal the ratio is preferably about 1500. One unit of D-AAO is defined as the amount of enzyme that consumes a xcexcmol of O2 per minute using cephalosporin C as substrate at pH 8.0 and 25xc2x0 C. One unit of catalase is defined as the amount of enzyme that decomposes 1 xcexcmol of hydrogen peroxide per minute at pH 7.0 and 25xc2x0 C.
d) The procedure of co-immobilisation. Several immobilisation protocols can be used. The one chosen in this invention is a modification of the method described by Cramer and Steckham (Tetrahedron, 45, 14645, 1997) for the co-immobilisation of L-xcex1-glycerolphosphate oxidase with catalase. Typically 100 mg of Eupergit(copyright) C250L are suspended in 1.5 ml of coupling buffer (1.0 M potassium phosphate buffer pH 8.0) in an Erlenmeyer flask. Then 10-40 U of D-AAO and 10-20 kU of catalase (Fluka, cat# 60634) are added slowly. The mixture is incubated for 16 h with gentle shaking. After the immobilisation procedure, the beads are separated by a glass frit and washed for several times using a 100 mM potassium phosphate buffer pH 7.0 at 4xc2x0 C.
Once the co-immobilisation is carried out, the enzymatic conversion of the compound III into compound IV is carried out in an aqueous solution of compound III containing from about 0.0016 to 0.004 moles and with less than 0.2 mg/ml of the heterocyclic thiol. This solution is obtained after passing the solution comprising 3-thiolated cephalosporin C (compound III) and remaining heterocyclic thiol used in the nucleophilic displacement of 3 acetoxy group of cephalosporin C through a column of a strong anion exchanger, such as Amberlite(copyright) IRA-400 (Rohm and Hass). The pH of the eluate is adjusted to pH about 6.5 to 8.0, preferably to pH 6.75, due to the instability of cephalosporanic compounds at basic pH values.
The solution comprising compound III as described above is fed into a bioreactor, containing wet Eupergit C250L with co-immobilised D-AAO/catalase, usually D-AAO from 20-40 U/g, and catalase, usually from 10-30 kU/g. The reaction temperature can be fixed from 15xc2x0 C. to 35xc2x0 C., and is normally fixed at 20xc2x0 C.
The molecular oxygen, needed for the oxidative deamination, is blown into solution by a bottom diffuser at a flow rate from 0.01 to 1 volume/volume of solution/minute, preferably at 0.1 vvm under a suitable mechanical stirring of about 400 rpm. This bioreactor design is preferred versus a percolation column containing the immobilised enzymes to avoid the diffusional problems of the molecular oxygen, which reduce the yield of compound IV. The pH is titrated to pH 6.75 by dosing a concentrated organic or inorganic base, preferably 3 M ammonia, by means of an autotitrator.
The conversion is controlled by HPLC and when the conversion of compound III is greater than 97%, the reaction is stopped and the solution filtered off. The time required for such conversion is of the order 0.5 to 3 hours, depending on the operating conditions, but usually approximately 1 hour.
Isolation of compound IV, when required, is carried out by decreasing the pH of the above solution to a pH of about 4.5 to 6.0, preferably 5.0 with the same base used during the enzymatic reaction, and loading into a column packed with the adsorptive resin Amberlite XAD-2. The elution of compound IV is carried out with water at a flow rate of 2-3 bed volumes per hour. Fractions containing the compound IV with a HPLC purity higher than 90-95% are pooled and lyophilised.
Compounds of formula IV are subsequently converted into compounds of formula I by enzymation with glutaryl-7-ACA acylase.
The process of the invention for preparing compounds of formula I from cephalosporin C may also be carried out in a single one pot reaction. In this case filtrate from an anion exchange column comprising compounds of formula III is enzymatically converted into compounds of formula I by an immobilised enzyme system comprising D-AAO and catalase in the presence of glutaryl-7-ACA acylase. Compounds of formula I have been prepared in this way with a HPLC of approximately 95%. The process is easy and efficient to carry out.
Using both processes (one-pot or two-steps) of the invention, 3-thiolated-7-ACA derivatives are easily and economically prepared. These compounds may by subsequent enzymation with penicillin G acylase for example, be used for the preparation of semisynthetic xcex2-lactam antibiotics. The xcex2-lactam antibiotics may include any one or more of cefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiam and ceftriaxone.
The following examples are meant to illustrate the invention without limitation as to its generality.
Examples 1 to 5 illustrates the preparation of 3-thiolated-7-ACA derivatives of formula III from cephalosporin C.
Examples 6 to 8 illustrate the enzymatic process for the preparation of a 3-thiolated xcex1-ketoadipyl-7-ACA derivatives of formula IV from 3-thiolated cephalosporin C derivatives of formula III.
Examples 9 to 11 illustrate the enzymatic process for the preparation of 3-thiolated-7-ACA derivatives (TXA) of formula I from 3-thiolated derivatives of formula III via the formation of stable xcex1-ketoadipyl-7-ACA derivatives of formula IV.
Examples 12 to 14 illustrate the enzymatic process for the preparation of 3-thiolated-7-ACA derivatives of formula I from 3-thiolated derivatives of formula III in a single step (one pot).